The Next-generation sequencing has experienced a period of rapid development since Roche invented the pyrosequencing method. However, with the development of high-throughput sequencing, high-throughput and sample preparation in low-cost gradually become key considerations in the sequencing field. Sample processing methods and automated devices based on various principles have been developed, including sample fragmentation, end-repairing and adaptor ligation to the nucleic acid molecules and a final library construction.
The sample fragmentation is mainly achieved by a physical method (such as ultrasonic shearing) or an enzymatic method (i.e., using non-specific endonuclease). The physical method is dominated by Covaris instrument which is based on Adaptive Focused Acoustic (AFA) technology. Under the isothermal condition, acoustic energy in a wavelength of 1 mm is focused on a sample geometrically by a spherical solid ultrasonic sensor at >400 kHz with, thereby guaranteeing the nucleic acid sample retained integrity, and achieving high recovery. Covaris instrument includes the economical M-series, S-series with single-tube and full-power, and E and L series with higher throughput. Although fragments obtained by the physical method are in good randomness, their throughput depends on Covaris instrument with high throughput, and such fragments obtained need to be subjected to end-repairing, adaptor ligation and various purifications subsequently. The enzymatic method includes NEB Next dsDNA Fragmentase developed by NEB company. This reagent can fragment double-stranded DNAs by randomly generating nicks on the double-stranded DNAs, followed by cutting the complementary double-stranded DNA chain with an enzyme which can recognize the nick sites. Although this reagent, with a good randomness, can be used in genomic DNAs, whole genome amplification products and PCR products, some artificial insertion and deletion of short fragment will be generated, and it is also unavoidable to proceed with end-repairing, adaptor ligation, PCR and corresponding purifications. Furthermore, the transposases fragmentation kit, led by the Nextera kit from Epicentra company (purchased by Illumina), may complete DNA fragmentation and adaptor ligation at the same time by means of the transposases, thereby reducing the time for sample preparation.
In view of the simplicity of the various operations, transposases fragmentation is undoubtedly far superior to other methods in terms of throughput and operation simplicity. However, such the fragmentation also has shortcomings. For example, transposition realized by the transposases depends on a specific 19 bp Me sequence. Therefore, though the transposases may ligate different adaptor sequences to a target sequence respectively at the 5′-terminal and the 3′-terminal by embedding two completely different adaptor sequences, the target sequence after fragmentation will symmetrically contain a Me sequence at each terminal thereof with a 9 nt gap formed between the target sequence and Me sequence due to the special function of the transposases. However, the identical Me sequences at two terminals of the target sequence will have an adverse influence on downstream technology applications. For example, when combing this adaptor ligation with the next-generation sequencing technology, the fact that the Me sequences located at two ends of the same strand of the target sequence are complementary to each other, will easily result in internal annealing within one single-stranded molecule, thus adversely contributing to combination with an anchoring primer.
There are few patents or other literatures so far reporting any molecular biology experimental method, which can extreme quickly and efficiently fragment a target sequence with transposases and correct the fragmented sequence to contain two completely different sequences at two terminal thereof.